Registration Dossier

Administrative data

Description of key information

The substance is considered to be not sensitizing to the skin of guinea pigs and humans. The test substance was also not considered a skin sensitizer in an in vitro Human Cell Activation Test (h-CLAT) and in an in chemico Direct Peptide Reactivity Assay (DPRA).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Basic data given, acceptable, well documented publication, no guideline and no GLP study.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The open epicutaneous test represents an animal bioassay conducted with guinea pigs and designated to generate quantitative data. The generation of skin sensitisation data includes a 3 step way. In the pretesting phase the primary irritation threshold concentration of the test substance is determined. The induction phase consists of a 3-week period of daily open applications. The final determination whether sensitisation has occurred or not takes place in the final challenge phase.
GLP compliance:
no
Type of study:
open epicutaneous test
Justification for non-LLNA method:
Information from a well documented publication is available and used in a weight of evidence approach.
Species:
guinea pig
Strain:
not specified
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 300-450 g
Route:
epicutaneous, open
Vehicle:
not specified
Concentration / amount:
100 %, 30 %, 10 %, 3 %, 1 % and 0.3 % / 21 x 0.1 mL
Day(s)/duration:
Day 0 - 20 / 3 weeks (4 weeks)
Adequacy of induction:
not specified
Route:
epicutaneous, open
Vehicle:
not specified
Concentration / amount:
0.025 mL
Day(s)/duration:
Days 21 - 35 / 24, 48, 72 hours
Adequacy of challenge:
other: minimal irritating concentration and some lower primary non irritating concentrations
No. of animals per dose:
Test group: At least 6 animals per dose
Control group: 10 animals per dose
Details on study design:
RANGE FINDING TESTS (Day 1):
Four guinea pigs were used for determination of the primary irritation concentration of the test substance so that non-irritating concentrations may be chosen for challenge.

MAIN STUDY
A. INDUCTION EXPOSURE (Day 0 - 20, epicutaneous)
- No. of exposures: 21 applications (daily for 3 weeks or 5 times weekly during 4 weeks)
- Exposure period: For 3 weeks (4 weeks)
- Control group: No treatment or 21 x 0.1 mL epicutaneous application of vehicle.
- Site: Clipped flank, on an area of 8 cm²
- Frequency of applications: Daily
- Readings: 24 hours after each application
- Duration: 3 weeks (4 weeks)
- Concentrations: if possible 100, 30, 10, 3, 1 and 0.3 %

B. CHALLENGE EXPOSURE (Days 21 - 35, epicutaneous)
- No. of exposures: 2
- Day(s) of challenge: 21 and 35
- Site: On the contralateral flank (2 cm²)
- Concentration: 0.025 mL of minimal irritating concentration and some lower primary non irritating concentrations
- Evaluation (hr after challenge): 24, 48 and 72 hours
Challenge controls:
no data
Positive control substance(s):
no
Positive control results:
no data
Key result
Reading:
other: Number of readings is not specified in the study
Group:
test group
Dose level:
10 %
No. with + reactions:
0
Remarks on result:
no indication of skin sensitisation
Interpretation of results:
GHS criteria not met
Conclusions:
In an open epicutaneous test (OET) groups of guinea pigs showed no sensitising effect at a concentration of 10 %.
Executive summary:

The skin sensitising potential of the test item was tested in a open epicutaneous test with guinea pigs. The test was conducted on groups of at least 6 male and female animals weighing 300 - 450 g. Daily applications were made for 3 weeks to a clipped 8 cm² area on the flank of each guinea pig. The test sites were not covered and the reactions were read 24 hours after each application. A total of 21 applications of 0.1 mL test material in an unspecified vehicle were made for 21 days. The 10 controls were either left untreated or treated with 0.1 mL test material in an unspecified vehicle for 21 days. At the challenge phase, both the test and control animals were treated at some lower primary non-irritating concentrations. At a test concentration of 10 % no sensitising effect were observed.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
see "Principles of method if other than guideline"
Principles of method if other than guideline:
The method is modified from Draize. In the Draize test sensitisation is induced by 10 intradermal injections of the test material at the ICC given over a 3 week period, whereas in this study the equivalent total dose was administered on one occasion as 4 intradermal injections, each 2.5 times the minimal irritating concentration (ICC: Injection challenge concentration).


GLP compliance:
not specified
Type of study:
other: modified Draize test
Justification for non-LLNA method:
Information from a publication is available and used in a weight of evidence approach.
Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: about 350 g
- Housing: In wire mesh cages in pairs of the same sex
- Diet: Animals were fed pelleted guinea pig diet, cabbage and hay ad libitum
- Water: ad libitum

Route:
intradermal
Vehicle:
not specified
Concentration / amount:
0.625% / 0.1 mL
Day(s)/duration:
Day 0 / 4 applications
No.:
#1
Route:
intradermal and epicutaneous
Vehicle:
not specified
Concentration / amount:
0.25 % and 20 % / 0.1 mL
Day(s)/duration:
Day 14
Adequacy of challenge:
other: ICC (0.25 %) and ACC (20 %)
No.:
#2
Route:
intradermal and epicutaneous
Vehicle:
not specified
Concentration / amount:
0.25 % and 20 % / 0.1 mL
Day(s)/duration:
Day 21
Adequacy of challenge:
other: ICC (0.25 %) and ACC (20 %)
No. of animals per dose:
10 (4 males, 6 females)
Details on study design:
PRELIMINARY IRRITATION TEST:
Intradermal injection: Four animals of the same sex and weighing about 450 g were each injected intradermally on the shaved flanks with 0.1 mL aliquots of a range of concentrations of the test substance in a suitable solvent. The reactions were examined for size (two largest diameters), erythema and oedema 24 h later and the concentration giving slight but perceptible irritation with no oedema was selected as the injection challenge concentration (ICC).
Topical application: Aliquots (0.1 mL) of a range of concentrations of the test substance in a suitable solvent were applied in small circular areas to the shaved flanks of 4 guinea pigs of the same sex and weighing about 450 g. The reactions were examined for erythema 24h later and the highest concentration which caused no irritation was selected as the application challenge concentration (AAC).

MAIN STUDY
A. INDUCTION EXPOSURE (Day 0, intradermal)
- No. of exposures: 4
- Site: Intradermally injections at 4 sites which overlie the 2 auxillary and 2 inguinal lymph nodes.
- Frequency of applications: 4
- Concentrations: 0.1 mL aliquots of the test substance at 2.5 times the ICC (0.25 %) = 0.625 %

B. CHALLENGE EXPOSURE (Day 14, intradermal and epicutaneous)
- No. of exposures: 1
- Site: Flanks (Intradermally in one flank and applied topically to the other flank)
- Concentrations: 0.1 mL aliquots of test substance at the respective ICC (0.25 %) and ACC (20 %).
- Evaluation (hr after challenge): 24

C. RECHALLENGE EXPOSURE (Day 21, intradermal and epicutaneous)
- No. of exposures: 1
- Control group: Control group included
- Site: Flanks (Intradermally in one flank and applied topically to the other flank)
- Concentrations: 0.1 mL aliquots of test substance at the respective ICC (0.25 %) and ACC (20 %).


OTHER:
In the absence of sensitisation reactions at first challenge the induction and challenge procedures were repeated, but this time a confirmatory challenge with controls was included irrespective of any apparent sensitization reactions at the previous challenge.
Challenge controls:
4 previously untreated animals of the same sex and similar weight to the test animals were treated intradermally and topically on opposite flanks with 0.1 mL aliquots of test substance at the ICC (0.25 %) and ACC (20 %) respectively.
Positive control substance(s):
no
Key result
Reading:
other: number of readings is not specified in the study
Group:
test group
Dose level:
0.25 %
No. with + reactions:
0
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
other: number of readings is not specified in the study
Group:
test group
Dose level:
20 %
No. with + reactions:
0
Remarks on result:
no indication of skin sensitisation
Interpretation of results:
GHS criteria not met
Conclusions:
The test substance showed no evidence of sensitisation under the test conditions chosen in this study.
Executive summary:

A modified Draize procedure was used to test 23 natural and 46 synthetic perfume ingredients, including the test substance, for their potential to induce allergic contact dermatitis in guinea pigs. After the performance of a preliminary irritation test to identify the minimal irritating (ICC, Injection challenge concentration) and the maximal non-irritating (ACC, Application challenge concentration) concentrations, 0.1 mL of the test substance at 2.5 times the ICC (0.625 %) was injected intradermally at 4 sites which overlie the 2 auxillary and 2 inguinal lymph nodes. 14 days later each animal was challenged intradermally in one flank and topically in the other with 0.1 mL aliquots of test substance at respective ICC (0.25 %) and ACC (20 %). 24 hours later the reactions were scored and apparent sensitisation reaction confirmed 7 days later by a second challenge with controls included. The test substance showed no evidence of sensitisation under the test conditions chosen in this study.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2016-03-07 to 2016-10-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD Guideline 442E (In Vitro Skin Sensitisation Human Cell Line Activation Test (h-CLAT))
Version / remarks:
Draft Guideline, December 2015
Deviations:
yes
Remarks:
The cytotoxicity measurement and estimation of the CV75 value of the dose finding assay was performed by XTT test instead of flow cytometry.
Principles of method if other than guideline:
In addition, the test guideline was performed according to the methods described in the following publications:
- Nukada Y., Ashikaga T., Miyazawa M., Hirota M., Sakaguchi H., Sasa H., Nishiyama N. (2012): Prediction of skin sensitization potency of chemicals by human Cell Line Activation Test (h-CLAT) and an attempt at classifying skin sensitization potency. Toxicol In Vitro. 2012 Oct;26(7):1150-60.
- Ashikaga T., Sakaguchi H., Sono S., Kosaka N., Ishikawa M., Nukada Y., Miyazawa M., Ito Y., Nishiyama N., Itagaki H. (2010): A comparative evaluation of in vitro skin sensitisation tests: the human cell-line activation test (h-CLAT) versus the local lymph node assay (LLNA). Altern Lab Anim. 2010 Aug;38(4):275-84.
GLP compliance:
yes (incl. certificate)
Remarks:
Envigo CRS GmbH, In den Leppsteinswiesen 19, 64380 Roßdorf
Type of study:
activation of dendritic cells
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 10300005
- Expiration date of the lot/batch: February 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
Details on study design:
Skin sensitisation (In vitro test system):
Cell line used: THP-1 cells (Human monocytic leukemia cell line) were purchased from ATCC, #TIB-202.
Justification for the Choice of cell line: THP-1 cells are used as surrogate for human myeloid dendritic cells and show enhanced CD86 and/or CD54 expression when treated with sensitizers.

Controls used:
- Medium control: Culture medium
- Positive control: DMSO, diluted with culture medium to an end concentration of 2 and 3 μg/mL DNCB.
- Solvent control for the test item: DMSO (final concentration for XTT cytotoxicity 0.2 - 0.5% and for h-CLAT 0.2%), used in the first and second XTT tests and in the first to third h-CLAT runs.
- Solvent control for the positive control (h-CLAT): DMSO (final concentration 0.2%)

Test substance preparation:
Since the test was initially not performed with the highest possible test item concentration, the dose range finder experiments (XTTs) and the h-CLAT runs were repeated with another vehicle (medium) and a higher test item concentration. On the day of the experiment (immediately prior to start) benzyl formate was solved in DMSO for the first and second XTT test and the first to third h-CLAT run or solved in medium for the first and second repetition of the XTT test and in the fourth and fifth h-CLAT run. The maximum soluble concentration of test item was 300 μg/mL (DMSO) and 1000 μg/mL (culture medium) as tested by two solubility tests. For the XTT test (dose finding assay) eight concentrations of the test item were analysed. Therefore, dilutions were prepared by 1:2 serial dilutions from the highest soluble concentration 300 μg/mL (DMSO) or 1000 μg/mL (culture medium).

THP-1 Cell Cultures:
Stocks of the THP-1 cell line are stored in liquid nitrogen in the cell bank of Envigo CRS GmbH (aliquots of cells in freezing medium at 1 × 106 to 2 × 10^6 cells/mL) allowing the repeated use of the same cell culture batch in experiments. Therefore, the parameters of the experiments remain similar, because of the reproducible characteristics of the cells. Thawed stock cultures will be propagated at 37 ± 1.5 °C in plastic flasks. The cells are sub-cultured twice weekly. The cell density should not exceed 1 × 10^6 cells/mL. The THP-1 cell suspension will be incubated at 37 ± 1.5 °C and 5.0 ± 0.5 % carbon dioxide atmosphere. Cells can be used up to three month after thawing (passage number should not exceed 30). The passage numbers of the used THP-1 cells in DMSO were 5 and 7 in the XTT assay and 7, 8 and 9 in the h-CLAT for runs 1, 2 and 3, respectively. The passage number of the used THP-1 cells in complete medium was 4 in the XTT assay and 5, 7 and 14 in the h-CLAT for runs 4, 5 and 6, respectively.

Preparation and Seeding of THP-1 Cells:
On the day of the cytotoxicity experiment (XTT) directly before the application of the test item, solvent and medium control, a volume of 100 μL with a cell density of 0.9 - 1 × 10^6 THP-1 cells/mL was seeded in each well of a 96-well flat bottom plate. For the main experiment (h-CLAT) 0.9 - 1 × 10^6 cells/well in a volume of 500 μL was seeded in a 24-well plate before the treatment.

Selection of concentrations (Dose Finding Assay, XXT Test):
The doses investigated in the main experiment (h-CLAT) were determined with two XTT test in DMSO and culture medium, respectively.
The XTT test is based on the cleavage of the yellow tetrazolium salt XTT [= (sodium 3'-(1-phenylaminocarbonyl) - (3,4 - tetrazolium) – bis - (4 – methoxy – 6 - nitro) - benzenesulfonic acid hydrate)] to form an orange water soluble formazan dye by dehydrogenase activity in active mitochondria. This method was first described 1988 by SCUDIERO et al. and improved in subsequent years by several other investigators.
Two independent cytotoxicity experiments (with DMSO as solvent) were performed with different cell cultures and on different days and two independent cytotoxicity experiments (with medium as solvent) were performed with different cell cultures to obtain a reliable CV75. The CV75 could not be determined therefore the highest soluble test item concentration was used to calculate the dose-range for the main experiment (h-CLAT).
After the cell seeding, 100 μL of the test item dilutions, the medium and solvent controls, respectively were added to the cells. All dose groups were tested 4 times. At the end of the incubation period of 24 ± 1 hours, the cell cultures were microscopically evaluated for morphological alterations.
At the end of the incubation period, 50 μL of the XTT labelling mixture were added to each well. The cells were incubated and subsequently transferred to a microplate reader (Versamax® Molecular Devices). The absorbance was measured at 450 nm (reference wavelength 690 nm). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

Calculation of the h-CLAT Test Doses:
Two independent cytotoxicity experiments were performed to obtain a reliable CV75. Since the CV75 could not be determined for the test item solved in culture medium or DMSO, a stock solution of the highest soluble test item concentration multiplied with 1.2 was prepared and seven further concentrations of the test item were prepared by serial 1:1.2 dilution.

Experimental Design and Procedures of h-CLAT:
The test item solved in DMSO was tested in two valid, independent runs. Due to a higher solubility of the test item in culture medium, additional three valid, independent runs were conducted.

Treatment of the Cells
For the test item exposure the highest dose solution calculated from the XTT assay was prepared corresponding to 1.2 × the highest concentration (in DMSO and culture medium) of the XTT tests instead of 1.2 × CV75, since no CV75 could be determined. Further 7 dilutions were prepared by individual 1:1.2 dilution. The dilutions were prepared freshly before each experiment. For the test item dissolved in DMSO, each solution was diluted with culture medium before application of the test solution to the cells to reach a final concentration of 0.2% (v/v) in the medium. Each volume (500 μL) of the dilutions of the test item, medium control, positive and DMSO control was added to the cells. The treated THP-1 cells will be incubated for 24 ± 1 hours. Each concentration of the test item, medium control, positive and DMSO control was tested at least in triplicates for the different staining (with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1).

Staining of the Cells:
The triplicates of each test item-treated and not test item treated cells were pooled and equally distributed into three sample tubes, collected by centrifugation (approx. 250 × g, 5 min) and then washed twice with approx. 2 mL of FACS buffer (PBS with 0.1% (w/v) BSA). Thereafter, the cells were centrifuged, re-suspended and blocked with 600 μL of blocking solution at 2 - 8 °C (on ice) for approx. 15 min. After blocking, the cells were centrifuged and the cell pellets were re-suspended in 100 μL FACS buffer. The cells were stained with FITClabelled anti-CD86, CD54 antibody or mouse IgG1 (isotype control). All solutions were kept light protected at 2 - 8 °C or on ice during the staining and analysis procedure. The cells were gently mixed by hand and incubated light protected for 30 ± 5 min. at 2 - 8 °C (on ice).

Sample Preparation for Measurement:
After staining with the antibodies, the cells were washed twice (2 - 8 °C) with 2 mL FACS buffer and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 minutes before the flow cytometry acquisition, 5 μL of a 7-AAD solution were added.

Flow Cytometry Acquisition:
Before using the flow cytometer, the device was calibrated with appropriate beads in accordance with the manufacturer’s instructions. The expression of cell surface antigens (CD54, CD86) was analyzed by flow cytometry. The FITC acquisition channel (FL-1) should be set for the optimal detection of the FITC fluorescence signal, and the 7-AAD acquisition channel (FL-3) should be set for the optimal detection of DNA-bound 7-AAD fluorescence signal.

Acquisition:
Dead cells were gated-out by staining with 7-AAD. Gating by FSC (forward scatter) and SSC (side scatter) was not done. A total of 10,000 living cells were analyzed. Mean fluorescence intensity (MFI) of viable cells and viability for each sample were used for analysis. The other tubes were acquired without changing the settings of the cytometer. The MFI was recorded for each condition. The relative fluorescence intensity (RFI) was not calculated, if the cell viability was less than 50 % (due to diffuse labelling of cytoplasmic structures that are generated due to cell membrane destruction).

Acceptance criteria:
The study is considered as valid, if the following criteria are met:
- Cell viability of medium control is adjusted to 100% and the cell viability of the DMSO control should be more than 90% in comparison to the medium control.
- In the positive control (DNCB), RFI values of both CD86 and CD54 should exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability should be > 50%; Culture medium, used in first and second repetition of the XTT tests and in the fourth and fifth h-CLAT runs.
- In the DMSO solvent control, RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).
- For medium and DMSO controls, the MFI ratio of CD86 and CD54 to isotype control should be > 105%.
- For the test item resulting in negative outcome, the cell viability at the 1.2 × CV75 should be less than 90%. (If the cell viability at the 1.2 × CV75 is more than 90% for a positive tested test item, the data will be acceptable. If 5 mg/mL in saline, 1 mg/mL in DMSO or the highest soluble dose will be used as the maximal test concentration instead of CV75-based dose, the data for test item are accepted independent by the cell viability).
- The cell viability of at least 4 doses in each experiment should be ≥50%.

Evaluation of results:
The test item is tested in 2 independent runs. If the RFI of CD86 is ≥ 150% or if the RFI of CD54 is ≥ 200% in both independent run data, the test item is considered to be a sensitizer. Otherwise it is considered to be a non-sensitizer. In case of different results in both runs, a third run has to be performed. If the RFI of CD86 is ≥ 150% at any dose in at least 2 of 3 independent run data, or if the RFI of CD54 is ≥ 200% in at least 2 of 3 independent run data, the test item is considered to be a sensitizer. Otherwise it is considered to be a non-sensitizer.
Positive control results:
The RFI values of the positive controls (DNCB) for CD86 and CD54 exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability was > 50%. Except the CD54 RFI value of the 2 μg/mL DNCB (positive control) treated cells of the fourth run did not exceed the positive criteria. However, this has no influence on the outcome of the study, since the RFI value of the 3 μg/mL DNCB treated cells exceeded positive criteria of both markers (CD86 ≥ 150% and CD54 ≥ 200%).
Key result
Remarks on result:
no indication of skin sensitisation
Remarks:
Please refer to "Any other information on results incl. tables" for detailed information.
Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of this study, the test item was not a skin sensitizer up to a concentration of 250 µg/mL dissolved in DMSO and up to a concentration of 578.7 µg/mL dissolved in culture medium.
Executive summary:

An in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the skin sensitizing potential of the test substance dissolved in DMSO and in culture medium (due to higher solubility), respectively when administered to THP-1 cells for 24 ± 1 hours. The dose for the main experiments was determined by two XTT test for each vehicle. In the first three experiments concentrations of the test substance (solved in DMSO) of 84, 100, 121, 145, 174, 208, 250 and 300 µg/mL were tested. In the fourth to sixth experiment the following concentrations of the test item (solved in culture medium) were tested: 334.9, 401.9, 4482.3, 578.7, 694.4, 833.3, 1000 and 1200 µg/mL.

The first h-CLAT run was not valid, since one acceptance criterion was not met. In the valid second and third run, the RFI of CD86 and CD54 was not ≥ 150% and ≥ 200%, respectively in any concentration of the test substance treated cells. The highest tested concentration (300 µg/L) was excluded from the evaluation, as phase separation was observed.

In the experiments with the test substance dissolved in culture medium, phase separation was observed in the four highest tested test item concentrations. Thus, these concentrations were excluded from the evaluation. Considering this, in two out of three runs the RFI of CD86 and CD54 was not ≥ 150% and ≥ 200%, respectively in at least one concentration of the test item treated cells. Therefore, the test item is considered to be not a sensitizer.

In all h-CLAT runs the DMSO solvent control, RFI values compared to the medium control of both CD86 and CD54 did not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%). Except in the first h-CLAT run the RFI values of the solvent control (DMSO) compared to the medium control of CD54 exceeded the positive criteria (CD54 ≥ 200%). The RFI values of the positive controls (DNCB) for CD86 and CD54 exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability was > 50%. Except the CD54 RFI value of the 2 μg/mL DNCB (positive control) treated cells of the fourth run did not exceed the positive criteria. However, this has no influence on the outcome of the study, since the RFI value of the 3 μg/mL DNCB treated cells exceeded positive criteria of both markers (CD86 ≥ 150% and CD54 ≥ 200%).

In conclusion, the test item benzyl formate was not a skin sensitizer up to a concentration of 250 µg/mL dissolved in DMSO and up to a concentration of 578.7 µg/mL dissolved in culture medium under the test conditions of this study.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Details on study design:
Skin sensitisation (In chemico test system) - Details on study design:
Acetonitrile solutions of the test item and the positive control were diluted with the Cysteine peptide so as to prepare solutions containing 0.5 mM Cysteine and 5 mM of either the test item or the positive control. For the co-elution control, buffer solution was used in place of the Cysteine stock solution.
Acetonitrile solutions of the test item and the positive control were diluted with the Lysine peptide so as to prepare solutions containing 0.5 mM Lysine and 25 mM of either the test item or the positive control. For the co-elution control, buffer solution was used in place of the Lysine stock solution.
Cinnamic aldehyde served as positive control. Additionally, stability controls and precision controls of both peptides were prepared.
The vials containing the samples were placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to initiation of the analysis run. The concentration of the Benzyl Formate and the associated positive control was quantified by HPLC using UV detection
Prior to the assay the solubility of the test substance was tested.
Positive control results:
The cysteine depletion was 73.0% and the lysine depletion was 56.3% after treatment with the psotive control.
Key result
Parameter:
other: peptide depletion (%)
Run / experiment:
#1 (cysteine depletion)
Value:
-0.536
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Parameter:
other: peptide depletion (%)
Run / experiment:
#2 lysine depletion
Value:
9.87
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY: All analytical acceptance criteria for each peptide run were met.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for reference control (i.e. samples containing only the peptide dissolved in the appropriate solvent): Yes (The mean cysteine peptide concentration was 0.509 mM and the mean lysine peptide concentration was 0.512 mM)
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes (CVs were less than 14.9% for cysteine peptide for the test item, positive and reference control)
Interpretation of results:
GHS criteria not met
Conclusions:
Based on the observed results and applying the prediction model according to Guideline, reactivity is classed as minimal and the DPRA prediction is negative. Therefore, the test item is a non-skin sensitizer.
Executive summary:

The reactivity of the test substance towards synthetic cysteine or lysine-containing peptides was evaluated in the DPRA according to OECD 442C.The test substance was incubated with synthetic peptides for a minimum of 22 hours at 25 °C and the remaining non-depleted peptide concentrations were determined by HPLC. The test substance was dissolved at a 100 mM concentration in acetonitrile. Three samples of the test substance were incubated with each peptide in ratios of 1:10 (Cysteine) or 1:50 (Lysine). The following results were obtained: The mean Cysteine-peptide depletion caused by the test substance was -0.536 %, the mean Lysine-peptide depletion caused by the test substance was 9.86%. No co-elution occurred during the test. Based on the observed results and applying the prediction model according to Guideline, reactivity is classed as minimal and the DPRA prediction is negative. Therefore, the test item is a non-skin sensitizer.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The skin sensitizing potential of the test item was determined in a weight of evidence approach consisting of two literature studies with guinea pigs, an in vitro Human Cell Line Activation Test (h-CLAT), an in chemico Direct Peptide Binding Assay (DPRA) and in human patch tests (see section 7.10.4). The results of these studies will be summarized in the following section. 

Animal studies

Open epicutaneous test of G. Klecak, 1985 (Curr Probl Dermatol 14 pg 152-71) The skin sensitising potential of the test item was tested in an open epicutaneous test with guinea pigs. The test was conducted on groups of at least 6 male and female animals weighing 300 - 450 g. Daily applications were made for 3 weeks to a clipped 8 cm² area on the flank of each guinea pig. The test sites were not covered and the reactions were read 24 hours after each application. A total of 21 applications of 0.1 mL test material in an unspecified vehicle were made for 21 days. The 10 controls were either left untreated or treated with 0.1 mL test material in an unspecified vehicle for 21 days. At the challenge phase, both the test and control animals were treated at some lower primary non-irritating concentrations. At a test concentration of 10 % no sensitising effect were observed. Modified Draize Test of Sharp, 1987 (Toxicology, 9: 261-271) A modified Draize procedure was used to test 23 natural and 46 synthetic perfume ingredients, including the test substance, for their potential to induce allergic contact dermatitis in guinea pigs. After the performance of a preliminary irritation test to identify the minimal irritating (ICC, Injection challenge concentration) and the maximal non-irritating (ACC, Application challenge concentration) concentrations, 0.1 mL of the test substance at 2.5 times the ICC (0.625 %) was injected intradermally at 4 sites which overlie the 2 auxillary and 2 inguinal lymph nodes. 14 days later each animal was challenged intradermally in one flank and topically in the other with 0.1 mL aliquots of test substance at respective ICC (0.25 %) and ACC (20 %). 24 hours later the reactions were scored and apparent sensitization reaction confirmed 7 days later by a second challenge with controls included. The test substance showed no evidence of sensitization under the test conditions chosen in this study.

 

In vitro test

Human Cell Line Activation Test (h-CLAT)

An in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the skin sensitizing potential of the test substance dissolved in DMSO and in culture medium (due to higher solubility), respectively when administered to THP-1 cells for 24 ± 1 hours. The dose for the main experiments was determined by two XTT test for each vehicle. In the first three experiments concentrations of the test substance (solved in DMSO) of 84, 100, 121, 145, 174, 208, 250 and 300 µg/mL were tested. In the fourth to sixth experiment the following concentrations of the test item (solved in culture medium) were tested: 334.9, 401.9, 4482.3, 578.7, 694.4, 833.3, 1000 and 1200 µg/mL.

The first h-CLAT run was not valid, since one acceptance criterion was not met. In the valid second and third run, the RFI of CD86 and CD54 was not ≥ 150% and ≥ 200%, respectively in any concentration of the test substance treated cells. The highest tested concentration (300 µg/L) was excluded from the evaluation, as phase separation was observed.

In the experiments with the test substance dissolved in culture medium, phase separation was observed in the four highest tested test item concentrations. Thus, these concentrations were excluded from the evaluation. Considering this, in two out of three runs the RFI of CD86 and CD54 was not ≥ 150% and ≥ 200%, respectively in at least one concentration of the test item treated cells. Therefore, the test item is considered to be not a sensitizer.

In all h-CLAT runs the DMSO solvent control, RFI values compared to the medium control of both CD86 and CD54 did not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%). Except in the first h-CLAT run the RFI values of the solvent control (DMSO) compared to the medium control of CD54 exceeded the positive criteria (CD54 ≥ 200%). The RFI values of the positive controls (DNCB) for CD86 and CD54 exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability was > 50%. Except the CD54 RFI value of the 2 μg/mL DNCB (positive control) treated cells of the fourth run did not exceed the positive criteria. However, this has no influence on the outcome of the study, since the RFI value of the 3 μg/mL DNCB treated cells exceeded positive criteria of both markers (CD86 ≥ 150% and CD54 ≥ 200%).

In conclusion, the test item benzyl formate was not a skin sensitizer up to a concentration of 250 µg/mL dissolved in DMSO and up to a concentration of 578.7 µg/mL dissolved in culture medium under the test conditions of this study.

 

In chemico test

Direct Peptide Reactivity Assay (DPRA)

The reactivity of the test substance towards synthetic cysteine or lysine-containing peptides was evaluated in the DPRA according to OECD 442C.The test substance was incubated with synthetic peptides for a minimum of 22 hours at 25 °C and the remaining non-depleted peptide concentrations were determined by HPLC. The mean Cysteine-peptide depletion caused by the test substance was -0.536 %, the mean Lysine-peptide depletion caused by the test substance was 9.86%. No co-elution occurred during the test. Based on the observed results and applying the prediction model according to Guideline, reactivity is classed as minimal and the DPRA prediction is negative. Therefore, the test item is a non-skin sensitizer.

 

Human patch tests - Kligmann, 1979 (RIFM Report, 72-8-140)

In a human maximisation study, the test material was applied under occlusion to the volar forearm of 25 healthy male subjects for five alternate-day 48 -hour periods. The patch sites were pre-treated for 24 hours with 5 % aqueous sodium lauryl sulfate under occlusion. Following a ten-day rest period challenge patches of all materials were applied under occlusion to fresh sites for 48 hours. Challenge applications were preceded by one-hour application of 10 % aqueous sodium lauryl sulfate under occlusion. The challenge sites were read on removal of the patch and 24 hours thereafter. No sensitisation was found after 48 and 72 hours.

Conclusion: 

Skin sensitizing studies on guinea pigs and human patch test from Kligmann showed no sensitizing potential under the test conditions chosen. The test substance was also not considered a skin sensitizer in an in vitro Human Cell Activation Test (h-CLAT) and in an in chemico Direct Peptide Binding Assay (DPRA) . Therefore, the substance is considered to be not classified according to Regulation (EC) No 1272/2008.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified for skin sensitisation under Regulation (EC) No 1272/2008, as amended for the ninth time in Regulation (EC) No 2016/1179.